This invention relates to the identification and use of Drosophila melanogaster as a model for screening of plant materials useful as psyhchostimulants.
Drugs that stimulate or depress CNS play an important role in human therapeutics. They act as anesthetics, analgesics, sedatives, psychostimulants, analeptics, antidepressants, anticonvulsants etc. and are used in the treatment of conditions such as narcolepsy, depression, hyperactivity disorders, epilepsy and drug addiction in human (Wood-Smith and Stewart, 1964, xe2x80x9cDrugs In Anesthetic Practice,xe2x80x9d Butterworth; Beckman, 1958, xe2x80x9cDrugs, Their Nature, Action and Use,xe2x80x9d W.B. Saunder, Green and Levy, 1976, xe2x80x9cDrug Misuse, Human Abuse,xe2x80x9d Dekker). Undesirable side effects and ineffectiveness of currently available CNS stimulants/depressants in many situations call for development of novel drugs. A variety of CNS active drugs have originated, and many of them are waiting to originate, from plants (Wood-Smith and Stewart, 1964, xe2x80x9cDrugs In Anesthetic Practice,xe2x80x9d Butterworth; Beckman, 1958, xe2x80x9cDrugs, Their Nature, Action and Use,xe2x80x9d W.B. Saunder; Green and Levy, 1976, xe2x80x9cDrug Misuse, Human Abuse,xe2x80x9d Dekker; Plotkin, 2000, xe2x80x9cMedicine Quest, In Search of Nature""s Healing Secrets,xe2x80x9d Viking; Gratzar, 2000, Nature 206:235-236). A reason why neuroactive compounds made by plants work on receptors in human brain is also known in the art (Lam et al, 1998, Nature 396:125-126). Keeping the above in view, the applicants used plant materials for screening neuroactive agents.
The applicants recently developed a Drosophila melanogaster model for in vivo whole organism based screening of CNS active agents (Sharma and Kumar, a method for screening of central nervous system active agents, U.S. patent application Ser. No. 09/789,525, filed concurrently herewith, the entire disclosure of which is incorporated herein by reference.In particular, the applicant showed that a change in the extent of time required by Sh5eag1 mutant flies to recover from ether anesthesia could be used as a criterion for rapid screening of CNS stimulant/depressant agents. In the present invention, this fruit fly has been used as a model for screening of plant materials.
The plant material screened and studied by the applicants was an extract obtained from Acorus calamus. Acorus calamus Linn. is a plant commonly used in Ayurvedic medicine. Its roots and rhizomes are used in various ailments, including many mental disorders, such as hysteria, insanity, insomnia, melancholia, neurasthenia, epilepsy, diarrhoea and asthma (Nadkarni, 1927, The Indian materia medica, Bombay, 26; Kirtikar and Basu, 1933, Indian medicinal plants, vol. IV, Lalit Mohan Basu, Allahabad, p.2626). The common Indian names of the plant are Ugargandha (Saskrit), ghor bach (Hindi), Bacha (Urdu), Wasa (Telugu) and Sweet flag (English).
Modern studies on the neuropharmacological actions of Acorus calamus have demonstrated CNS activity in roots and rhizomes, not leaves, of the plant (Bhakuni et al, 1988, Ind. J. Exp. Biol. 26:883-904; Bhakuni et al, 1969, Ind. J. Exp. Biol., 7:250-262). Mainly, CNS depressant actions of Acorus calamus have been reported (Zanoli et al, 1998, Phytother, Res. 12:S114-S116; Panchal et al, 1989, Ind. J. Exp. Biol. 27:561-567; Dandiya and Sharma, 1962, Ind. J. Med. Res. 50:46-60; Chopra et al, 1954, Ind. J. Med. Res. 42:381384; Menon and Dandiya, 1966, J. Pharm, Pharmac. 19:170-175; Dhalla et al, 1961, J. Pharmaceut. Sci. 50:580-582; Dhalla and Bhattacharya, 1968, Arch. Int. Pharmacodyn. 172:356-365; Madan et al, 1960; CXXIV:201-211; Dandiya and Menon, 1965, Life Sci. 4:1635-1641).
The active principles identified are asarone and xcex2-asarone, obtained from roots and rhizomes after steam distillation of volatile oil. Various workers have mainly characterized as having CNS depressant actions such as sedative, tranquilizing, hypnosis potentiation, anticonvulsant, spontaneous motor activity antagonizing, amphetamine induced hyperactivity, antagonizing and analgesic activities to roots and rhizomes of Acorus calamus. 
A CNS stimulant action of the oil from the plant has been reported by Chopra et al, 1954, md. J. Med. Res., 42:381-384). The authors have however given no proof in support of their statement. It is possible that the generalized convulsions induced by xcex2-asarone might have made these workers ascibe a stimulant property to Acorus calamus (Panchal and Venkatkrishna-Bhatt, 1989, Ind. J. Exp. Biol. 27:561-567; Dandiya and Sharma, 1962, Ind. J. Med. Res., 50:46-60). In addition, based on the studies related to the effect of asarone on the leamed behaviour of the rats it has been suggested that the substance may cause a stimulant action at a higher dose and depression at a lower dosage (Dandiya and Sharma, 1962, Ind .J. Med. Res., 50:46-60).
Thus, in the prior art, only roots and rhizomes, not leaves, of Acorus calamus are known to possess drug-like activities. Also, the known neuroactivities, of the plant are limited to actions such as sedation, tranquilizing, hypnosis potentiation and convulsions.
Therefore, the extract obtained from the leaves of this plant was screened for CNS stimulant/depressant activity. The Applicants, to their surprise found that the extract works as an excellent CNS stimulant/depressant.
The main object of the invention is to provide a method whereby plant materials can be screened to test their efficacy as CNS depressant/stimulants.
Another object of the invention is to provide a method for treatment of poisoning, narcolepsy, depression, hyperactivity disorders, alcohol/drug addiction etc. in humans.
The invention provides a method for screening of plant materials as CNS stimulant/depressant using Drosophila melanogaster as a model.
Accordingly, the invention provides a novel method whereby plant materials can be screened for their efficacy as CNS stimulant/depressants. The extract of several plants including Acorus calamus was tested for CNS stimulation/depression activity in the mutant fruit fly Drosophila melanogaster Sh5eag1.
The invention provides a method for the detection of the analeptic and psychostimulant properties of extract obtained from Acorus calamus, using Drosophila melanogaster Sh5eag1 said method comprising the steps of:
(a) preparing acetone and methanol extracts of leaves of the plant Acorus calamus, 
(b) treating Sh5eag1 Drosophila melanogaster mutant flies in the media selected from normal fly food, normal food mixed with phenobarbital, normal food mixed with ethanol, normal food mixed with the alcoholic sample prepared above, normal food mixed with both the alcoholic sample prepared above and phenobarbital and normal food mixed with both the alcoholic sample of step (a) and ethanol,
(c) subjecting differently treated flies to ether anesthesia and then determining extent of time required by them to recover,
(d) observing spontaneous locomotor activity in the differently treated flies and increase in the spontaneous locomotor activity is indicative of analeptic activity of extract of Acorus calamus against ethanol,
(e) Observing the recovery time following ether and chloroform anesthesia in flies not pretreated with phenobarbital wherein quick recovery of the flies is indicative of analeptic activity of the extract against phenobarbital,
(f) Observing the recovery time in flies anesthetized with ether and pretreated with phenobarbital, wherein quick recovery of the flies is indicative of analeptic activity of the extract,
(g) observing the spontaneous locomotor activity of the flies under the influence of ethanol, wherein quick recovery of the flies is indicative of analeptic activity of the extract and
(h) observing the spontaneous locomotor activity of the flies not under the influence of ethanol, wherein quick recovery of the flies is indicative of the psychostimulant activity of the extract.
In an embodiment, the extract of Acorus calamus is directly used or used as a source of an analeptic, a psychostimulant, an antidepressant, an antabuse and a somnolytic.
In another embodiment, the extract of Acorus calamus is directly used or used as a source of a therapeutic agent to treat depressant poisoning, narcolepsy, depression, hyperactivity disorders and alcohol/drug addiction in humans.
Using a fruit fly model, more than 20 plant species were tested for neuroactive agents. Extracts of the plant Acorus calamus were found to decrease the time taken by flies to recover from anesthesia, a state of severe CNS depression, produced by diethyl ether. Further experiments with one of the Acorus calamus extracts showed a similar effect on flies anesthetized with chloroform. Also, the extract suppressed delay in recovery from ether anesthesia caused by CNS depressant drug phenobarbital. In addition, the inhibition in flies"" spontaneous locomotor activity caused by ethanol, an another CNS depressant, was also suppressed. The above results therefore demonstrate that the natural substance screened has a property akin to known analeptics, drugs and antagonize the action of CNS depressants. The plant extract was also found to increase spontaneous locomotor activity, an expression of CNS activity status, in normal flies. This further suggests that the agent also possess a property similar to known psychostimulants. Accordingly, the present invention provides a substance from Acorus calamus plant with analeptic and psychostimulant properties which comprises use of a Drosophila model for in vivo drug screening, testing of a large number of plant extracts from various plant species and finding in the plant Acorus calamus a substance with analeptic and psychostimulant properties.
The invention is illustrated by the following examples, which are provided to illustrate the invention and should not be construed as limitation on the inventive concept herein.